Surface monofunctionalization of protein nanostructures will enable precise topological control over the protein-templated assembly of nanoscale motifs, however, this remains a formidable challenge. Here we demonstrated a novel strategy for this purpose with a protein nanocage, virus-based nanoparticle (VNP) of simian virus 40 as a model system. By simultaneously incorporating a function modality (cysteine) and a purification modality (polyhistidine tag) into the building block (VP1) of VNPs through rational design and genetic engineering, the monofunctionalized cysteine-VNPs are readily obtained through a routine affinity chromatography in virtue of the purification modality of polyhistidine tag, after the coassembly of the functional VPI and the nonfunctional VP1 at an optimal ratio. This strategy has proved to be highly efficient in constructing monofunctionalized protein nanostructures as highlighted by the monofunctionalized-VNP-guided Au/QD-VNP nanostructures. These nanostructures could be utilized in a wide range of disciplines, including basic biological research, novel nanostructures, and nanodevices fabrication, etc.